Methods for producing transgenic plants with enhanced resistance and decreased uptake of heavy metals

ABSTRACT

The present invention relates to a method of producing transformants with enhanced resistance and decreased uptake of heavy metals, and a plant transformed with a P type ATPase ZntA gene that pumps out heavy metals from the cells. The transformants show better growth than wild type in environment contaminated with heavy metals and have lower heavy metal contents than wild type plants. Therefore, this method of transforming plants with ZntA or biologically active ZntA—like heavy metal pumping ATPases can be useful for developing plants for phytoremediation and also for a safe crop that has resistance to heavy metals and low heavy metal contents.

This application is a 371 of PCT/KR02/00605 filed April 4, 2002.

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The present invention relates to a method of producing transformantswith enhanced heavy metal resistance. More particularly, the presentinvention relates to transgenic plants that have an improved growth butdecreased heavy metal contents when grown in environment contaminatedwith heavy metals, thus this method can be used for developing plantsfor phytoremediation and also for developing safe crops.

(b) Description of the Related Art

Heavy metals are major environmental toxicants, which cause reactiveoxidation species generation, DNA damage, and enzyme inactivation bybinding to active sites of enzymes in cells.

Contamination of the environment with heavy metals has increaseddrastically due to industrialization. By the early 1990s, the worldwideannual release had reached 22,000 tons of cadmium, 954,000 tons ofcopper, 796,000 tons of lead, and 1,372,000 tons of zinc (Alloway B J &Ayres D C (1993) Principles of environmental pollution. Chapman andHall, London). The soils contaminated with heavy metal inhibit normalplant growth and cause contamination of foodstuffs. Many heavy metalsare very toxic to human health and carcinogenic at low concentrations.Therefore removal of heavy metals from the environment is an urgentissue.

Studies for removing heavy metals from soil are very activelyprogressing worldwide. Traditional methods of dealing with soilcontaminants include physical and chemical approaches, such as theremoval and burial of the contaminated soil, isolation of thecontaminated area, fixation (chemical processing of the soil toimmobilize the metals), and leaching using an acid or alkali solution(Salt D E, Blaylock M, Kumar N P B A, Viatcheslav D, Ensley B D, et al.(1995). Phytoremediation: a novel strategy for the removal of toxicmetals from the environment using plants. Bio-Technology 13,468–74;Raskin I, Smith R D, Salt D E. (1997) Phytoremediation of metals: usingplants to remove pollutants from the environment. Curr. Opin.Biotechnol. 8, 221–6). These methods, however, are costly andenergy-intensive processes.

Phytoremediation has recently been proposed as a low-cost,environment-friendly way to remove heavy metals from contaminated soils,and is a relatively new technology for cleanup of contaminated soil thatuses general plants, specially bred plants, or transgenic plants toaccumulate, remove, or detoxify environmental contaminants. Thephytoremediation technology is divided into phytoextraction,rhizofiltration, and phytostabilization.

Phytoextraction is a method using metal-accumulating plants to extractmetals from soil into the harvestable parts of the plants;rhizofiltration is a method using plant roots to remove contaminantsfrom polluted aqueous streams; and phytostabilization is thestabilization of contaminants such as toxic metals in soils to preventtheir entry into ground water, also with plants (Salt et al.,Biotechnology 13(5): 468–474, 1995).

Examples of phytoremediation are methods using the plants of Larreatridentate species that are particularly directed at the decontaminationof soils containing copper, nickel, and cadmium (U.S. Pat. No.5,927,005), and a method using Brassicaceae family (Baker et al., NewPhytol. 127:61–68, 1994).

In addition, phytoremediation using transgenic plants that are generatedby introducing genes having resistant activity for heavy metals havebeen attempted. Examples of heavy metal resistant genes are CAX2(Calcium exchanger 2), cytochrome P450 2E1, NtCBP4 (Nicotiana tabacumcalmodulin-binding protein), GSHII (glutathione synthetase), merB(organomercurial lyase), and MRT polypeptide (metal-regulatedtransporter polypeptide).

CAX2 (Calcium exchanger 2), isolated from Arabidopsis thaliana,accumulates heavy metals including cadmium and manganese in plants(Hirschi et al., Plant Physiol. 124:125–134, 2000). Cytochrome P450 2E1uptakes and decomposes organic compounds such as trichloroethylene (DotySL et al., Proc. Natl. Acad. Sci. USA 97:6287–6291, 2000). Nicotianatabacum transformed with NtCBP4 has resistant activity for nickel (Araziet al., Plant J. 20:171–182, 1999), GSHII accumulates cadmium (Liang etal., Plant Physiol. 119:73–80,1999), merB detoxifies organic mercury(Bizily et al., Proc. Natl. Acad. Sci. USA 96:6808–6813, 1999), and MRTpolypeptide removes heavy metals including cadmium, zinc, and manganesefrom contaminated soil (U.S. Pat. No. 5,846,821).

However, the transgenic plants generated by introducing theabove-mentioned genes have limitations in growth due to accumulation ofheavy metals, and they can produce contaminated fruits and crops, whengrown in contaminated soil. Therefore, there is a need for plants thathave a lower uptake of heavy metals than the wild type, and thatmaintain healthy growth even in an environment contaminated with heavymetals.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a gene, when expressed inplants, that confers heavy metal resistance and that can inhibitaccumulation of heavy metals.

It is a further object of the invention to provide a recombinant vectorharboring a heavy metal resistant gene.

It is a further object of the invention to provide a method forproducing transformants that have heavy metal resistance and thataccumulate less heavy metals than wild type plants.

It is a further object of the invention to provide transformants thathave heavy metal resistance and that accumulate less heavy metals thanwild type plants.

It is a further object of the invention to provide a method oftransforming a polluted area into an environmentally friendly space.

To accomplish the aforementioned objects, the invention provides arecombinant vector containing a coding sequence for a heavymetal-transporting P type ATPase, wherein the coding sequence isoperably linked to and under the regulatory control of aplant-expressible transcription and translation regulatory sequence.

Also, the invention provides a transgenic plant, or parts thereof, eachtransformed with a recombinant vector.

Also, the invention provides a transgenic plant cell.

Also, the invention provides a transgenic plant, stably transformed witha recombinant vector.

Also, the invention provides a recombinant vector comprising a codingsequence for a heavy metal-transporting P type ATPase, ZntA of SEQ IDNO: 1;

-   -   wherein the coding sequence is operably linked to and under the        regulatory control of a plant-expressible transcription and        translation regulatory sequence; and

wherein the ZntA contains an approximately 100 amino acid residueN-terminal extension domain, a first transmembrane spanning domain, asecond transmembrane spanning domain containing a putative cationchannel motif CPX domain, a third transmembrane spanning domain, a firstcytoplasmic domain, a second cytoplasmic domain, and a C-terminal domain

Also, the invention provides a recombinant vector comprising a codingsequence for a heavy metal-transporting P type ATPase, ZntA wherein thecoding sequence is operably linked to and under the regulatory controlof a plant-expressible transcription and translation regulatory;

wherein the ZntA contains an approximately 100 amino acid residueN-terminal extension domain, a first transmembrane spanning domain, asecond transmembrane spanning domain containing a putative cationchannel motif CPX domain, a third transmembrane spanning domain, a firstcytoplasmic domain, a second cytoplasmic domain, and a C-terminaldomain; and

wherein each of the domains of the coding sequence shares at least about50% homology with a same domain of SEQ ID NO:1.

Also, the invention provides a method of producing a transgenic plantwith enhanced resistance to heavy metals comprising:

(a) preparing an expression construct comprising a sequence encoding aheavy metal-transporting P type ATPase, operably linked to and under theregulatory control of a plant-expressible transcription and translationregulatory sequence;

(b) preparing a recombinant vector harboring the expression construct;and

(c) introducing the expression construct of the recombinant vector intoa plant cell or plant tissue to produce a transgenic plant cell ortransgenic plant tissue

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the map of the recombinant vector pEZG.

FIG. 2 shows plasma membrane localization of ZntA protein expressed inArabidopsis protoplasts.

FIG. 3 is a Western blot photograph showing membrane localization ofZntA protein expressed in Arabidopsis protoplast.

FIG. 4 represents the map of recombinant vector PBI121/zntA.

FIG. 5 is a Northern blot photograph showing expression of zntA mRNA inArabidopsis.

FIG. 6 shows the enhanced growth of zntA-transgenic plants over that ofwild type in a medium containing lead.

FIG. 7 shows the enhanced growth of zntA-transgenic plants over that ofwild type in a medium containing cadmium.

FIG. 8 is a graph showing the weight of zntA-transgenic pants cultivatedin media containing heavy metals.

FIG. 9 is a graph showing the chlorophyll contents of zntA-transgenicand wild type plants, grown in media containing heavy metals.

FIG. 10 is a graph showing the heavy metal contents of zntA-transgenicand wild type plants, grown in media containing heavy metals.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used herein, the term “P type ATPase” refers to a transporter thattransports a specific material by using energy from ATP hydrolysis andthat forms a phosphorylated intermediate. More particularly, the P typeATPase is a heavy metal-transporting ATPase. The heavy metal is a metalelement having a specific gravity over 4 including arsenic (As),antimony (Sb), lead (Pb), mercury (Hg), cadmium (Cd), chrome, tin (Sn),zinc, barium (Ba), nickel (Ni), bismuth (Bi), cobalt (Co), manganese(Mn), iron (Fe), copper (Cu), and vanadium (V).

ZntA is a P type ATPase of E. coli (Rensing C, Mitra B, Rosen B P.(1997) Proc. Natl. Acad. Sci. USA. 94,14326–31; Sharma, R., Rensing, C.,Rosen, B. P., Mitra, B. (2000) J Biol. Chem. 275,3873–8) which pumpsPb(II)/Cd(II)/Zn(II) across the plasma membrane.

P-type ATPases typically have 2 large cytoplasmic domains and 6transmembrane domains. ZntA has similar domains, and in addition, 2 moretransmembrane helixes at N-terminus and N-terminal extension of about100 amino acids containing CXXC motif. The first large cytoplasmicdomain of ZntA is about 145 amino acid long and involved in hydrolysisof phosphointermediate, and the second large cytoplasmic domain is 280amino acid long and has a phosphorylation motif. We denote the 4transmembrane helixes of the N-terminal side as the first transmembranespanning domain. The 2 transmembrane helixes between the 2 largecytoplasmic domains is denoted as the second transmembrane spanningdomain. This domain contains a putative cation channel motif CPX domain.The transmembrane helixes between the second large cytoplasmic domainand the c-terminus is denoted as the third transmembrane spanningdomain. The cytoplasmic domain following the third transmembranespanning domain is denoted as the C-terminal domain of ZntA.

The term “homology” refers to the sequence similarity between 2 DNA orprotein molecules. “Biologically active ZntA-like heavy metal pumpingATPases” are coded by DNA sequences which have at least 50% homology toZntA, and have heavy metal pumping activity. Biologically activeZntA-like heavy metal pumping ATPases include zinc-transporting ATPase(NC_(—)000913), zinc-transporting ATPase (NC_(—)002655), heavymetal-transporting ATPase (NC_(—)003198), P-type ATPase family(NC_(—)003197), cation transporting P-type ATPase from Mycobacteriumlepraed (GenBank #Z46257), and many others.

A “heavy metal resistance protein” is a protein capable of mediatingresistance to at least one heavy metal, including, but not limited to,lead, cadmium, and zinc. An example of a heavy metal resistance proteinis ZntA protein of SEQ ID NO:2.

The term “plant-expressible” means that the coding sequence is operablylinked to and under the regulatory control of a transcription andtranslation regulatory sequence that can be efficiently expressed byplant cells, tissues, parts and whole plants.

“Plant-expressible transcriptional and translational regulatorysequences” are those which can function in plants, plant tissues, plantparts and plant cells to effect the transcriptional and translationalexpression of the target sequence with which they are associated.Included are 5′ sequences of a target sequence to be expressed, whichqualitatively control gene expression (turn gene expression on or off inresponse to environmental signals such as light, or in a tissue-specificmanner); and quantitative regulatory sequences which advantageouslyincrease the level of downstream gene expression. An example of asequence motif that serves as a translational control sequence is thatof the ribosome binding site sequence. Polyadenylation signals areexamples of transcription regulatory sequences positioned downstream ofa target sequence, and there are several that are well known in the artof plant molecular biology.

A “transgenic plant” is one that has been genetically modified, unlikethe wild type plants. Transgenic plants typically express heterologousDNA sequences, which confer the plants with characters different fromthat of wild type plants. As specifically exemplified herein, atransgenic plant is genetically modified to contain and express at leastone heterologous DNA sequence that is operably linked to and under theregulatory control of transcriptional control sequences which functionin plant cells or tissue, or in whole plants.

The present invention provides a plant-expressible expression constructcontaining a coding sequence for a heavy metal-transporting ATPaseprotein. The coding sequence is operably linked to and under theregulatory control of a plant-expressible transcription and translationregulatory sequence. The heavy metals include arsenic (As), antimony(Sb), lead (Pb), mercury (Hg), cadmium (Cd), chrome, tin (Sn), zinc,barium (Ba), nickel (Ni), bismuth (Bi), cobalt (Co), manganese (Mn),iron (Fe), copper (Cu) and vanadium (V).

The expression construct includes a promoter, a heavy metal-transportingP type ATPase gene, and a transcriptional terminator. The suitableplant-expressible promoters include the 35S or 19S promoters ofCauliflower Mosaic Virus; the nos (nopaline synthase), ocs (octopinesynthase), or mas (mannopine synthase) promoters of Agrobacteriumtumefaciens Ti plasmids; and others known to the art.

The heavy metal-transporting ATPase gene of the present inventionprefers genes encoding ZntA (SEQ ID NO:1) or biologically activeZntA-like heavy metal pumping ATPase genes, which have at least 50%homology to ZntA, and which code for proteins with heavy metal pumpingactivities.

The heavy metal-transporting ATPase gene of the present invention alsoprefers DNA sequences containing an approximately 100 amino acid residueN-terminal extension domain, a first transmembrane spanning domain, asecond transmembrane spanning domain containing a putative cationchannel motif CPX domain, a third transmembrane spanning domain, a firstcytoplasmic domain, a second cytoplasmic domain, and a C-terminal domainof ZntA, or DNA sequences which share at least 50% homology withabovementioned domains of the biologically active ZntA-like heavy metalpumping ATPase genes.

The expression construct of the present invention may further contain amarker allowing selection of transformants in the plant cell or showinga localization of a target protein. The examples of a marker are genescarrying resistance to an antibiotic such as kanamycin, hygromycin,gentamicin, and bleomycin; and genes coding GUS (α-glucuronidase), CAT(chloramphenicol acetyltransferase), luciferase, and GFP (greenfluorescent protein). The marker allows for selection of successfullytransformed plant cells growing in a medium containing certainantibiotics because they will carry the expression construct with theresistance gene to the antibiotic.

Also, the invention provides a recombinant vector comprising theexpression construct. The recombinant vector comprises a backbone of thecommon vector and the expression construct. The common vector ispreferably selected from the group consisting of pROKII, pBI76, pET21,pSK(+), pLSAGPT, pBI121, and PGEM. Examples of the prepared recombinantvector are PBI121/zntA and pEZG. PBI121/zntA comprises a backbone ofPBI121, CMV 35S promoter, zntA gene, and nopaline synthase terminator;and pEZG comprises a backbone of pUC, CMV 35S promoter, zntA gene, greenfluorescence protein, and nopaline synthase terminator.

Also, the present invention provides a transformant containing theexpression construct. The transformant contains a DNA sequence encodinga heavy metal-transporting P type ATPase, wherein the coding sequence isoperably linked to and under the regulatory control of a transcriptionand translation regulatory sequence.

The transformant is preferably a plant, and more preferably a plant,parts thereof, and plant cell. The plant parts include a seed. Theplants are herbaceous plants and trees, and they include floweringplants, garden plants, an onion, a carrot, a cucumber, an olive tree, asweet potato, a potato, a cabbage, a radish, lettuce, broccoli,Nicotiana tabacum, Petunia hybrida, a sunflower, Brassica juncea, turf,Arabidopsis thaliana, Brassica campestris, Betula platyphylla, a poplar,a hybrid poplar, and Betula schmidtii.

Techniques for generating transformants are well known. An example isAgrobacterium tumefaciens-mediated DNA transfer. Preferably, recombinantA. tumefaciens generated by electroporation, micro-particle injection,or with a gene gun can be used.

In addition, the invention provides a method of producing a transgenicplant with enhanced resistance to heavy metals, comprising:

(a) preparing an expression construct comprising a plant-expressiblesequence encoding a heavy metal-transporting P type ATPase, operablylinked to and under the regulatory control of a transcription andtranslation regulatory sequence;

(b) preparing a recombinant vector harboring the expression construct;and

(c) introducing the expression construct of the recombinant vector intoa plant cell or plant tissue to produce a transgenic plant cell ortransgenic plant tissue.

The method of producing a transgenic plant further comprises a step: (d)regenerating a transgenic plant from the transgenic plant cell ortransgenic plant tissue of step (c).

In the present invention, ZntA protein was expressed in the plasmamembrane (FIGS. 2 and 3). Moreover, zntA-transgenic Arabidopsis plantsshowed enhanced resistance to lead and cadmium, and the content of leadand cadmium was lower than in a wild-type plant.

Therefore, zntA-transgenic plants or plants transformed with a geneencoding biologically active ZntA-like heavy metal pumping ATPases cangrow in an environment contaminated with heavy metals, and thistechnique can be useful for generating crop plants with decreased uptakeof harmful heavy metals. Since harmful heavy metals can be introducedinto farmland inadvertently, for example, due to the yellow sandphenomenon or by natural disaster, heavy metal pumping transgenic cropplants can be a safe choice for health-concerned consumers.

The following examples are provided for illustrative purposes and arenot intended to limit the scope of the invention as claimed herein. Anyvariations in the exemplified compositions and methods which occur tothe skilled artisan are intended to fall within the scope of the presentinvention.

EXAMPLE 1 Isolation of ZntA Gene

Escherichia coli K-12 was obtained from the Korean Collection for TypeCultures of the Korea Research Institute of Bioscience andBiotechnology, and a zntA gene was cloned.

zntA was isolated by PCR using genomic DNA of Escherichia coli K-12strain as a template. PCR was performed with a primer set of SEQ IDNO:4, SEQ ID NO:3, and 2.2 kb of PCR product, and zntA of SEQ ID NO:1was obtained. The sequence of the PCR product was aualyzed and the PCRproduct was cloned into a pGEM-T easy vector to produce pCEM-T/zntA.

EXAMPLE 2 Expression of ZntA Protein

A zntA gene was, introduced into Arabidopsis protoplasts, andlocalization of ZntA protein was investigated.

(2-1) Preparation of Arabidopsis Protoplasts

Arabidopsis protoplasts were prepared as described (Abel S, Theologis A(i 994) Transient transformation of Arabidopsis leaf protoplasts: aversatile experimental system to study gene expression. Plant J. 5,421–7).

Seeds of Arabidopsis were placed into an antiseptic solution (distilledwater: chlorox: 0.05% triton X-100=3:2:2), shaken for 20–30 seconds, andincubated at room temperature for 5–10 mins. The seeds were then rinsedfive times with distilled water.

The Arabidopsis seeds were incubated in 100 ml of a liquid solution(Murashige & Skoog medium; MSMO, pH 5.7–5.8) containing vitamins,Duchefa 4.4 g/L, sucrose 20 g/L, MES (2-(N-Morpholino) Ethanesulfonicacid, Sigma) 0.5 g/L, while agitating at 120 rpm under a 16/8 hr(light/dark) cycle, at 22° C. for 2–3 weeks.

The 2–3 week-old whole plants were chopped with a razor blade to 5–10mm² pieces. These leaf fragments were transferred to an enzyme solution(1% cellulase R-10, 0.25% marcerozyme R-10, 0.5 M mannitol, 10 mM MES, 1mM CaCl₂, 5 mM β-mercaptoethanol, and 0.1% BSA, pH 5.7–5.8),vacuum-infiltrated for 10 min, and then incubated in the dark at 22° C.for 5 hours with gentle agitation at 50–75 rpm. The released protoplastswere filtered through a 100 μm mesh (Sigma S0770, USA), purified using a21% sucrose gradient by centrifugation at 730 rpm for 10 min, and thensuspended in 20 ml of W5 solution (154 mm NaCl, 125 mM CaCl₂, 5 mM KCl,5 mM glucose, and 1.5 mM MES, pH 5.6) and centrifuged again at 530 rpmfor 6 min. The pellected protoplasts were re-suspended in W5 solutionand kept on ice.

(2-2) Preparation of Vector

pGEM-T/zntA DNA was cut with BamHI restriction enzyme and zntA geneswere extracted (QIAGEN Gel extraction kit). The zntA genes were placedunder the control of a Cauliflower Mosaic Virus 35S promoter, fused withand then inserted into a pUC-GFP vector containing Green FluorescentProtein (GFP) and nopaline synthase terminator (NOS), to thereby producepEZG.

(2-3) Preparation of Vector for H⁺ Pumping Gene

A hydrogen ion pump gene of Arabidopsis, AHA2 cDNA (Gene Bank: P19456),was amplified by PCR. Primers for PCR were polynucleotides of SEQ IDNO:4 and SEQ ID NO:5. PCR conditions were as follows: 94° C., 30sec->45° C., 30 sec->72° C., 1 min, 50 cycles. The PCR product wasobtained as AHA2 cDNA.

A DsRed vector (Clontech, Inc.) was treated with BgIII/NotI restrictionenzyme and DsRed was obtained. The DsRed was inserted into the openedsmGFP vector with a BamHI/EcI136II restriction enzyme to 326RFP. Inaddition, AHA2 cDNA was inserted at XmaI of the 326RFP vector and326RFP/AHA2 was prepared.

(2-4) Introduction of pEZG or 326RFP/AHA2 Into Protoplast

pEZG and 326RFP/AHA2 were introduced to the protoplasts prepared byEXAMPLE (2-1), and expression of foreign genes was confirmed.

The protoplast was centrifuged at 500 rpm for 5 min, and 5×10⁶/ml of theprotoplast were suspended in a MaMg solution (400 mM mannitol, 15 mMMgCl₂, 5 mM MES-KOH, pH 5.6). 300 μl of the suspension solution wasmixed with 10 μg of pEZG and 326RFP/AHA2 respectively, which was thenwas added to 300 μl of PEG (400 mM mannitol, 100 mM Ca(NO₃)₂, 40% PEG6000), and stored at RT for 30 min. The mixture was washed with 5 ml ofW5 solution, centrifuged at 500 rpm for 3 min, and a pellet wasobtained. The pellet was washed with 2 ml of W5 solution and incubatedin the dark at 22–25° C. After 24 hr, expression of GFP protein wasmonitored and images were captured with a cooled charge-coupled devicecamera using a Zeiss Axioplan fluorescence microscope. The filter setsused for the GFP were XF116 (exciter, 474AF20; dichroic, 500DRLP;emitter, 510AF23) (Omega, Inc., Brattleboro, Vt.). Data were thenprocessed using Adobe (Mountain View, Calif.) Photoshop software.

FIG. 2 shows a localization of ZntA protein fused with GFP inprotoplasts transformed with pEZG and 326RFP/AHA2, respectively. “a” iscontrol, “b” is AHA2 protein expressed in 326RFP/AHA2, “c” is ZntAprotein expressed in pEZG, and “d” is an overlapped picture of “b” and“c”. ZntA fused with GFP shows a green color due to GFP, and AHA2 fusedwith DsRed shows a red color due to DsRed.

In FIG. 2, ZntA fused with GFP was localized at the plasma membrane inArabidopsis protoplasts.

In addition, membrane and cytosol fractions were isolated fromArabidopsis protoplasts, and Western Blot was preformed using a GFPantibody as a probe. FIG. 3 is a Western Blot photograph, wherein “WT-C”is cytosol of wild-type Arabidopsis protoplasts, “WT-M” is membrane ofwild-type Arabidopsis protoplasts, “ZntA-C” is cytosol of Arabidopsisprotoplasts transformed with pEZG, and “ZntA-M” is membrane ofArabidopsis protoplasts transformed with pEZG. In FIG. 3, the GFPantibody cross-reacted only with membrane proteins extracted fromArabidopsis protoplasts transformed with pEZG, confirming that ZntAprotein was expressed in membrane.

EXAMPLE 3 Preparation of Transgenic Plants Expressing ZntA Protein.

(3-1) Arabidopsis

Arabidopsis plants were grown at 4° C. for 2 days, then they were grownwith a 16/8 hr (light/dark) photoperiod, at 22° C./18° C. for 3–4 weeksuntil flower stalks were differentiated. The 1^(st) flower stalk wasremoved, and the 2^(nd) flower stalk was used for transformation.

(3-2) pBI121/ZntA Vector

A zntA gene was inserted into the expression vector for the plant,preparing pBI121 and pBI121/zntA.

A GUS gene of pBI121 was removed by digesting with SmaI and EcI136IIrestriction enzymes, and a zntA gene prepared from the pGEM-T/zntA wasinserted to pBI121, thereby preparing a pBI121/zntA vector (FIG. 4).

(3-3) Preparation of Transgenic Plants

pBI121/zntA vector DNA was isolated with a prep-kit (Qiagen) andintroduced to agrobacterium using electroporation. The agrobacterium(KCTC 10270BP) was cultured in YEP media (yeast extract 10 g, NaCl 5 g,pepton 10 g, pH 7.5) until index of O.D. reached 0.8–1.0. The culturesolution was centrifuged, cells were collected and suspended in MS media(Murashige & Skoog medium, 4.3 g/L, Duchefa) containing 5% sucrose, andSilwet L-77 (LEHLE SEEDS, USA) was added as a final concentration of0.01% just before transformation. For plant transformation, pBI121/zntAwas introduced into the Agrobacterium LBA4404 strain, which was thenused to transform Arabidopsis by a dipping method (Clough S J, and BentA F (1988), Floral dip: a simplified method for Agrobacterium-mediatedtransformation of Arabidopsis thaliana. Plant J. 16, 735–743).

EXAMPLE 4 Selection of Transformants

For selection of plant transformed with zntA genes, plants were grown insolid Murashige-Skoog (MS) medium containing kanamycin (50 mg/l). T2 orT3 generation seeds were used for the tests. Also, a pBI121 vector wasintroduced to Arabidopsis and transformants (pBI121 plants) wereselected. Seeds were obtained from wild-type Arabidopsis, pBI121 plants,and pBI121/zntA plants, respectively.

To test the ZntA expression level, total RNA was isolated fromkanamycin-selected T2 plants and used for Northern Blot analysis. TotalRNA was extracted from Arabidopsis plants grown on the 1/2 MS (Murashige& Skoog medium, 2.15 g/L, Duchefa)-agar media for 3 weeks. SubsequentRNA preparation and northern hybridization followed the establishedmethod (Sambrook et al. (2001) Molecular Cloning: A laboratory manual(Third

Edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)with slight modifications.

The plant materials were frozen in liquid nitrogen and homogenized withmortars and pestles. 1 ml of TRIzol reagent (Life technology, USA) per100 mg of tissue was added to the sample and after 5 min incubation atRT, 0.2 ml of chloroform per 1 ml of TRIzol reagent was added. Aftercentrifugation at 10,000 g for 10 min at 4° C., the aqueous phase wastaken and precipitated with 0.5 ml of isopropyl alcohol per 1 ml ofTRIzol reagent and quantified by UV spectroscopy. Total RNA wasseparated in a formaldehyde-containing agarose gel and then transferredonto a nylon membrane. After UV crosslinking, hybridization was carriedout in a modified Church buffer (7% (w/v) SDS, 0.5 M sodium phosphate(pH 7.2), 1 mM EDTA (pH 7.0)) at 68° C. overnight, with ³²P-labeled zntAprobes. Membranes were washed once for 10 min in 1×SSC, 0.1% SDS at roomtemperature, and twice for 10 min in 0.5×SSC, 0.1% SDS at 68° C. Themembrane was exposed to a phosphorimager screen (Fuji film) or x-rayfilm (Kodak). The mRNA expression levels were analyzed by the Mac-BASimage-reader program. FIG. 5 is a Northern Blot photograph showingexpression of zntA mRNA in Arabidopsis. Transcription of zntA RNA wasnot observed in wild-type Arabidopsis and pBI121 plants, but it wasobserved in pBI121/zntA plants. EF1-a is constitutively expressed inplants and its even levels indicated that the same amount of RNA wasused for different samples.

EXAMPLE 5

Heavy Metals Resistance of Plant Transformed With ZntA Gene

Wild-type Arabidopsis plants and pBI121/zntA plants were grown in 1/2MS-agar media for 2 weeks and transferred 1/2 MS-liquid media containing70 μM cadmium or 0.7 mM lead. After 2 weeks, growth, weight, and heavymetal contents were measured.

(5-1) Growth of Plants

FIG. 6 shows the growth of wild-type and pBI121/zntA Arabidopsis plantsgrown in a medium containing lead. FIG. 7 shows wild-type andpBI121/zntA Arabidopsis plants grown in a medium containing cadmium.“WT” is wild-type Arabidopsis, “1” to “4” are pBI121/zntA plants. InFIGS. 6 and 7, pBI121/zntA plants grew better than the wild-type plants;their leaves were broader, greener, and their fresh weights were higherthan those of the wild types. These results indicate that the expressionof ZntA confers Pb(II)- and Cd(II)-resistance to the transgenic plants.

(5-2) Measurement of Biomass

Wild type and pBI121/zntA Arabidopsis plants were grown in 1/2 MS-agarmedia for 2 weeks and then transferred to 1/2 MS-liquid media supportedby small gravel with or without Cd (II) or Pb (II). After growing for anadditional 2 weeks, the plants were harvested. They were washed in anice-cold 1 mM tartarate solution and blot-dried. The weight of the wildtype and pBI121/zntA Arabidopsis plants were measured.

FIG. 8 a is a graph showing the weight of wild type and pBI121/zntAplants grown in a medium containing lead, and FIG. 8 b is a graphshowing the weight of wild type and pBI121/zntA plants grown in a mediumcontaining cadmium. The weight of pBI121/zntA plants was higher thanthat of the wild-type plants. These results indicate that plantsexpressing ZntA protein can grow better than wild type in soilcontaminated with heavy metals.

(5-3) Measurement of Chlorophyll Contents

For determination of chlorophyll contents, the leaves were harvested andextracted with 95% ethanol for 20 min at 80° C. Absorbance at 664 nm and648 nm were measured and then the chlorophyll A and B contents werecalculated as described (Oh S A, Park J H, Lee G I, Paek K H, Park S K,Nam H G (1997) Identification of three genetic loci controlling leafsenescence in Arabidopsis thaliana. Plant J. 12, 527–35).

FIG. 9 a is a graph showing the chlorophyll contents of wild type andzntA-transgenic plants grown in a medium containing lead, and FIG. 9 bis a graph showing the chlorophyll contents of wild type andzntA-transgenic plants grown in a medium containing cadmium. Thechlorophyll contents of zntA-transgenic plants were higher than those ofthe wild types.

(5-4) Measurement of the Heavy Metal Contents

We measured the content of Pb and Cd in control and ZntA overexpressingplants grown in media containing heavy metals. pBI121/zntA plants werecollected, weighed, and digested with 65% HNO₃ at 200° C., overnight.Digested samples were diluted with 0.5 N HNO₃ and analyzed using anatomic absorption spectrometer (AAS; SpectrAA-800, Varian).

FIG. 10 is a graph showing the heavy metal contents of wild type andzntA-transgenic plants grown in media containing heavy metals. FIG. 10 ais the lead contents, and 10 b is the cadmium contents. Pb content ofpBI121/zntA plants varied between the lines, but it was consistentlylower than that of the wild type. Cd content in transgenic lines 1 and 3was lower than that in the control.

Thus, plants transformed with zntA or other biologically activeZntA-like heavy metal pumping ATPases can be grown in soil contaminatedwith heavy metals and have less uptake of heavy metals than wild typeplants. Since growing plants can hold contaminated soil and therebyreduce erosion of the soil, and since the zntA-transgenic plants cangrow better than wild type plants in soil contaminated by heavy metals,they can reduce migration of pollutants from the polluted area, therebyreducing contamination of groundwater by the pollutants. The presentinvention can also be applied to crop plants to produce low heavymetal-containing safe crop plants.

1. A method of producing a transformed plant or part thereof comprisingtransforming the plant or part thereof with a recombinant vectorcomprising a nucleic acid sequence encoding ZntA having the amino acidsequence of SEQ ID NO:2, wherein the nucleic acid sequence is operablylinked to a plant-expressible regulatory sequence.
 2. The methodaccording to claim 1, wherein the nucleic acid sequence comprises SEQ IDNO:
 1. 3. The method according to claim 1, wherein the recombinantvector is PBI121/zntA.
 4. A transgenic plant or part thereof withenhanced resistance to a heavy metal produced by the method of claim 1.5. The transgenic plant or part thereof according to claim 4, whereinthe heavy metal is at least one selected from the group consisting ofarsenic, antimony, lead, mercury, cadmium, chrome, tin, zinc, barium,nickel, bismuth, cobalt, manganese, iron, copper, and vanadium.
 6. Atransgenic plant cell with enhanced resistance to a heavy metal,transformed with the a recombinant vector comprising a nucleic addsequence encoding ZntA having the amino acid sequence of SEQ ID NO:2. 7.The transgenic plant cell according to claim 6, wherein the heavy metalis at least one selected from the group consisting of arsenic, antimony,lead, mercury, cadmium, chrome, tin, zinc, barium, nickel, bismuth,cobalt, manganese, iron, copper, and vanadium.
 8. A transgenic plantwith enhanced resistance to a heavy metal, stably transformed with arecombinant vector comprising a nucleic acid sequence encoding ZntAhaving the amino acid sequence of SEQ ID NO:2.
 9. The transgenic plantaccording to claim 8, wherein the heavy metal is at least one selectedfrom the group consisting of arsenic, antimony, lead, mercury, cadmium,chrome, tin, zinc, barium, nickel, bismuth, cobalt, manganese, iron,copper, and vanadium.
 10. A transgenic plant or part thereof withenhanced resistance to a heavy metal, each transformed with arecombinant vector comprising the nucleic acid sequence of SEQ ID NO:1operably linked to a plant-expressible regulatory sequence.
 11. Thetransgenic plant or part thereof according to claim 10, wherein theheavy metal is at least one selected from the group consisting ofarsenic, antimony, Lead, mercury, cadmium, chrome, tin, zinc, barium,nickel, bismuth, cobalt manganese, iron, copper, and vanadium.
 12. Atransgenic plant cell with enhanced resistance to a heavy metal,transformed with a recombinant vector comprising the nucleic acidsequence of SEQ ID NO:1 operably linked to a plant-expressibleregulatory sequence.
 13. The transgenic plant cell according to claim12, wherein the heavy metal is at least one selected from the groupconsisting of arsenic, antimony, lead, mercury, cadmium, chrome, tin,zinc, barium, nickel, bismuth, cobalt, manganese, iron, copper, andvanadium.
 14. A transgenic plant with enhanced resistance to a heavymetal, stably transformed with a recombinant vector comprising thenucleic acid sequence of SEQ ID NO:1 operably linked to aplant-expressible regulatory sequence.
 15. The transgenic plantaccording to claim 14, wherein the heavy metal is at least one selectedfrom the group consisting of arsenic, antimony, lead, mercury, cadmium,chrome, tin, zinc, barium, nickel, bismuth, cobalt, manganese, iron,copper, and vanadium.
 16. A method of producing a transgenic plant cellor transgenic plant tissue with enhanced resistance to heavy metalscomprising: (a) preparing an expression construct comprising a nucleicacid sequence encoding ZntA protein having the amino add sequence of SEQID NO: 2, operably linked to a plant-expressible regulatory sequence;(b) preparing a recombinant vector harboring the expression construct,and (c) introducing the expression construct of the recombinant vectorinto a plant cell or plant tissue to produce a transgenic plant cell ortransgenic plant tissue.
 17. The method according to claim 16, whereinthe heavy metal is at least one selected from the group consisting ofarsenic, antimony, lead, mercury, cadmium, chrome, tin, zinc, barium,nickel, bismuth, cobalt, manganese, iron, copper, and vanadium.
 18. Themethod according to claim 16, further comprising the step of:regenerating a transgenic plant from the transgenic plant cell ortransgenic plant tissue of step (c).